Antibody drug conjugates (ADCs) have received considerable attention due to promising clinical results, with a therapeutic recently receiving FDA approval. (Alley et al., Curr. Opin. Chem. Bio. 2010, 14, 529-37; Katz et al., Clin. Cancer Res. 2011, 17 6428-36) The specific cell targeting, as directed by the antibody, allows for the use of extremely toxic small molecules, with the targeting resulting in reduced off target side effects. These properties have led to the possibility of resurrecting small molecule therapeutics, which have been discarded from the development pipeline due to unfavorable toxicities or pharmacokinetics. To date the predominant method of attaching drugs to antibodies has been via a covalent linkage; some of which can be cleaved intracellularly through chemical or enzymatic reactions. (Alley et al., Curr. Opin. Chem. Bio. 2010, 14, 529-37) The specificity afforded by antibodies (and antibody fragments) have also resulted in their use for the targeted delivery of liposomes, dendrimers, metallic nanoparticles and nanoparticles of non-metallic composition. (Cheng et al., Expert Opin. Drug Deliv. 2010, 7, 461-78; New et al., Mol. Pharm. 2012, 9, 374-81; Chattopadhyay et al., Mol. Pharm. 2012, 9, 2168-79; Tivnan et al., PLoS One 2012, 7, e38129) These species have been exploited for the delivery of therapeutic and imaging agents either through non-covalent encapsulation (Lehtinen et al., PLoS One 2012, 7, e41410) or covalent attachment. (New et al., Mol. Pharm. 2012, 9, 374-81) In some cases, e.g., gold nanoparticles and quantum dots, the nanoparticle may also be the therapeutic or imaging agent. (Melancon et al., Biomaterials 2011, 32, 7600-8; Ruan et al., Biomaterials 2012, 33, 7093-102)
Chemically self-assembled antibody nanorings (CSANs) can be formed through the oligomerization of dimeric dihydrofolate reductase antiCD3 single chain variable fragment (scFv) fusion proteins (DHFR2antiCD3) with a bis-methotrexate (bisMTX) ligand (FIG. 1). (Li et al., Angew. Chem. Int. Ed. 2008, 47, 10179-82; Li et al. J. Am. Chem. Soc. 2010, 132, 17247-57). AntiCD3 CSANs are endocytosed upon binding to CD3+ T cells via a clathrin dependent mechanism, in a manner similar to that of the parental antiCD3 monoclonal antibody (mAb), UCHT-1. (Li et al. J. Am. Chem. Soc. 2010, 132, 17247-57) It was hypothesized that antiCD3 CSANs could be used for the delivery of dyes and drugs to T-leukemia cells for imaging and therapeutic purposes (FIG. 2). Control over ring size, and hence the valency of the displayed scFv, is possible through modification of the length and composition of the amino acid linker between the DHFR proteins: a 13 amino acid linker (13DHFR2antiCD3) results in a mixture of monomeric and dimeric species; a shorter single glycine linker (1DHFR2antiCD3) leads to predominantly octameric rings. (Li et al., Angew. Chem. Int. Ed. 2008, 47, 10179-82; Li et al., J. Am. Chem. Soc. 2010, 132, 17247-57; Carlson et al., J. Am. Chem. Soc. 2006, 128, 7630-8). This offers the possibility of controlling the number of therapeutic molecules delivered by the CSANs, as well as tuning the affinity for the targeted cells. Further the modular nature of the CSAN construct allows changes in size, valency, and loading of cargo. As these properties can modulate cellular endocytosis. (Muzykantov et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2379-84; Wiewrodt et al., Blood 2002, 99, 912-22) CSANs are a useful construct for which to study these effects.
Despite the ability of the antiCD3 CSANs to selectively bind and undergo endocytosis, little is known about the intracellular behavior of these unique nanoparticles, or their ability to carry cargoes into cells. In part to address this issue, a unique trivalent bisMTX ligand was designed and synthesized. Molecules, such as dyes, radiolabels or drugs, can be attached. The attachment of a fluorophore has allowed verification of the ability of CSANs to act as a carrier. Also because the fluorescence of the dyes is quenched upon release from the CSANs, the intracellular stability of the CSANs was monitored under a variety of conditions. Further, CSANs can be disassembled intracellularly by DHFR inhibitors. To probe the potential for endosomal escape, while bisMTX is non-toxic to HPB-MLT (a T leukemia cancer cell line) antiCD3 CSAN mediated delivery resulted in an increase in cytotoxicity of at least 13-fold. Taken together, these results demonstrate that T-leukemia cells endocytose intact antiCD3 CSANs, which have a significant life-time in the cell, and that upon disassembly bisMTX is released into the cytoplasm.